Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM

Ryanodine receptors (RyRs) are large Ca ²⁺ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs were identified in mammals, disfunction of which was associated with a series of life-threatening diseases. Advances in structural studies of RyRs are limited by the need for large amounts of native tissue or eukaryotic cell cultures. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification starting from isolated membranes to cryo-EM grade protein is completed within four hours on the bench and produces protein usable for cryo-EM as we show by solving the structures of rabbit RyR1 and bovine and mouse RyR2 solubilized in detergent, reconstituted into lipid nanodiscs or liposomes. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in the cases where the amount of starting material is limited.